Due to the expanding health consciousness of people around the world, there has been an increasing demand for vitamin C. Also contributing to the demand for ascorbic acid is its widespread use as an antioxidant for preserving food. One approach for satisfying this demand is to achieve increased production of 2-KLG, an intermediate in the production of ascorbic acid. The intermediate, 2-KLG, can be easily converted to ascorbic acid through acid or base catalyzed cyclization. It also has a greater stability and shelf life than ascorbic acid. Therefore, rather than producing ascorbic acid directly, it is more practical to stockpile 2-KLG for subsequent conversion to ascorbic acid.
A number of species of a first group of microorganisms, Erwinia, Acetobacter, and Gluconobacter, can produce 2,5-DKG from D-glucose. A second group of microorganisms from the coryneform group of bacteria (Corynebacterium, Brevibacterium, and Arthrobacter) as well as species of Micrococcus, Staphylococcus, Pseudomonas, Bacillus, and Citrobacter are capable of converting 2,5-DKG, produced by a microorganism of the first group, to 2-KLG. This cofermentation of appropriate microorganisms to produce 2-KLG was simplified by combining the relevant traits of both the Erwinia sp. and the Corynebacterium sp. in a single microorganism (Anderson et al., Science 23: 144 (1985)). This was accomplished by identifying the 2,5-DKG reductase in the Corynebacterium sp. that converts 2,5-DKG into 2-YLG. The gene for this reductase was then cloned and expressed in Erwinia herbicola, a bacterium of the family Enterobacteriaceae that converts D-glucose into 2,5-DKG in a single fermentation. The resulting recombinant bacterial strain, with 2,5-DKG reductase as the pivotal enzyme, was able to convert D-glucose into 2-KLG in a single-fermentation process (Lazarus et al. Fourth ASM Conf. Genet. Molec. Biol. Indust. Microorg., 187-193 (1989)).
Improving the catalytic efficiency of 2,5-DKG reductase, in the single-fermentation process, is a significant way to increase the production of 2-KLG. Also, a purified 2,5-DKG reductase A with increased catalytic activity could be used in an in vitro process for the conversion of 2,5-DKG to 2-KLG. For example, such a process would permit continuous production of 2-KLG through immobilization of the purified enzyme on a solid support.
According to the Michaelis-Menten scheme set out below, the
      E    ⁢          +      ⇀        ⁢    S    ⁢      ↽    Km    ⁢      ES    ⁢          →      kcat        ⁢          E      +      P      efficiency of an enzymatic reaction can be measured by two kinetic parameters, kcat and Km. The catalytic rate constant, kcat, also known as the turnover number, is a measure of the breakdown of the enzyme-substrate (ES) complex. It also represents the maximum number of substrate molecules (S) converted to product (P) via an ES complex per active site of the enzyme (E) per unit time. Vmax is the maximal velocity or rate of the enzyme catalyzed reaction when the enzyme is saturated with substrate. Therefore, Vmax is constant at saturating substrate concentration and remains unchanged with any increase in substrate concentration. The kcat at saturating substrate concentrations is related to Vmax and the total enzyme concentration, [ET], by the following equation: Vmax=kcat [ET]. The Michaelis constant, Km, is equal to the dissociation constant of the ES complex. Therefore, Km is a measure of the strength of the ES complex. In a comparison of Km's, a lower Km represents a complex with a stronger, more favorable binding, while a higher Km represents a complex with a weaker, less favorable binding. The ratio, kcat/Km, called the specificity constant, represents the specificity of an enzyme for a substrate, i.e., the catalytic efficiency per enzyme molecule for a substrate. The larger the specificity constant, the more preferred the substrate is by the enzyme.
Impressive yields of 2-KLG have been achieved with a Corynebacterium 2,5-DKG reductase (2,5-DKG reductase A, also known as 2,5-DKG reductase II) (Anderson et al., Science 230: 144 (1985); Miller et al., J. Biol. Chem. 262: 9016 (1987)) expressed in appropriate host strains (2,5-DKG producers) such as Erwinia sp. These results have been achieved despite 2,5-DKG reductase A having a low specificity constant for 2,5-DKG. Since Corynebacterium does not naturally encounter 2,5-DKG, it is not surprising that this compound is a poor substrate for 2,5-DKG reductase A.
This low specificity constant for 2,5-DKG reductase A is in contrast to a second, homologous Corynebacterium 2,5-DKG reductase (2,5-DKG reductase B, also known as 2,5-DKG reductase I) that has a greater specificity constant for 2,5-DKG (Sonoyama and Kobayashi, J. Ferment. Technol. 65: 311 (1987)). In addition, both 2,5-DKG reductases are homologous to several known aldose and keto-reductases that have greater specificity constants towards their known substrates. Such findings indicate that the active site of 2,5-DKG reductase A is not optimally configured for the catalytic conversion of 2,5-DKG to 2-KLG. Therefore, it appears that in order to optimize 2,5-DKG reductase A specific activity in the single-fermentation process, amino acid substitutions by site-directed mutagenesis must be made to the enzyme's active site.
In addition to improving an enzyme's kinetic parameters, site-directed mutagenesis can increase structural stability by amino acid substitutions, deletions, or insertions. The following are examples of structurally stabilizing mutations. The introduction of new disulfide bonds to create covalent crosslinks between different parts of a protein has been used to improve the thermal stability of bacteriophage T4 lysozyme (Matsumura et al., Nature 342:291-293 (1989)), bacteriophage λ repressor (Sauer et al., Biochem. 25:5992-5998 (1986)), E. coli dihydrofolate reductase (Villafranca et al., Biochem. 26:2182 (1987)), and subtilisin BPN′ (Pantoliano et al., Biochem. 26:2077-2082 (1987)). There is a computer program (Pabo et al., Biochem. 25:5987-5991 (1986)) that permits efficient scanning of the crystallographically determined three-dimensional structure of a protein to suggest those sites where insertion of two cysteines might lead to disulfide bonds. Such bonds would not disrupt the larger-scale conformation, while stabilizing the local conformation.
Amino acid substitutions of alanine for glycine in the α-helix have been shown to increase the thermal stability of the bacteriophage λ repressor (Hecht et al., Proteins Struct. Funct. Genet. 1:43-46 (1986)) and the neutral protease from Bacillus stearothermophilus (Imanaka et al., Nature 324:695-697 (1986)). An increase in the melting temperature, Tm, for bacteriophage T4 lysozyme was accomplished by the two amino acid substitutions of proline for alanine and alanine for glycine (Matthews et al., Proc. Nat. Acad. Sci. USA 84:6663-6667 (1987)). Replacement of amino acids in the hydrophobic core of a protein with aromatic residues such as tyrosine, especially at positions near preexisting clusters of aromatic side chains, has been shown to promote thermal stability in kanamycin nucleotidyl transferase (Liao et al., Biochem. 83:576-580 (1986)) and bacteriophage λ repressor (Hecht et al., Biochem. 81:5685-5689 (1984)).
Transcriptional and translational control sequences in expression vectors are the key elements required for the high level production of proteins in bacteria. The E. coli Trp, bacteriophage λPL, E. coli lac UV5, and the Trp-lacUV5 fusion (Tac) promoters are among the most frequently used prokaryotic promoters (de Boer et al., Proc. Nat. Acad. Sci. USA 80: 21-25 (1983); Sambrook et al., Molecular Cloning, Cold Spring Harbor Press (1989); Remaut et al., Gene 15:81-93 (1981)). There is no way to determine whether a particular protein will be highly expressed upon induction of transcription from these promoters. The translational efficiency of the message, mRNA stability, and the protein's intrinsic stability are the major factors in high-level expression. Therefore, whenever a protein undergoes mutagenesis it is always possible its expression level will be affected.
Site-directed mutagenesis, using synthetic DNA oligonucleotides having the desired sequence, permits substitution, deletion, or insertion of selected nucleotides within a DNA sequence encoding a protein of interest. Recombinant DNA procedures are used to introduce the desired mutation by substituting the synthetic sequence for the target sequence. Development of plasmids containing an origin of replication derived from a filamentous bacteriophage (Vieira and Messing, Methods in Enzymology 153: 3 (1987)) permits cloning of fragments into single stranded forms of plasmids capable of autonomous replication. Use of such plasmids eliminates the arduous task of subcloning DNA fragments from plasmids to filamentous bacteriophage vectors. Kits for carrying out site-directed mutagenesis are commercially available.
Mutants of 2,5-DKG reductase A having characteristics which vary from the native enzyme would be useful. In particular, mutants having improved catalytic activity, enhanced thermal stability, and increased expression levels would be useful to extend the commercial utility of the enzyme.
Unfortunately, unless proteins share regions of substantial sequence or structural homology, it is not possible to generalize among proteins to predict, based on a beneficial mutation of one protein, precisely where the sequence encoding another protein should be changed to improve the performance of that protein. Therefore, it is necessary to undertake an analysis of the precise structural and functional features of the particular protein to be altered. This determines which amino acids to alter to produce a desired result, such as increased catalytic activity, thermostability, or expression.
The present invention provides mutated forms of enzymatically active prokaryotic 2,5-DKG reductase A. Analysis of the structure of 2,5-DKG reductase A to select alterations encoding the enzyme to enhance stability, expression, and/or activity of resulting mutants was undertaken. Site-directed mutagenesis of the sequence encoding the enzyme was designed to produce the mutants.